Sleep is of central importance to neurobiology because to understand how the brain works, we will have to decipher the mechanisms and functions of sleep. The function(s) of sleep remain unknown and the humoral and neural mechanisms of sleep are incompletely understood. Most people intuitively recognize that sleep increases after sleep loss or during the course of an infection. There is much evidence that those sleep responses, as well as physiological sleep, are regulated, in part, by humoral mechanisms. We hypothesize that tumor necrosis factor alpha (TNF-alpha) is one of the key substances in sleep regulation. This hypothesis is based on studies showing: 1) TNF-alpha induces non-rapid eye movement sleep (NREMS); 2) inhibition of TNF-alpha inhibits spontaneous sleep and sleep responses induced by sleep loss or bacterial products; 3) TNF mRNA and TNF brain levels correlate with sleep propensity; 4) in humans, circulating TNF levels correlate with electroencephalogram slow-wave activity and increase after sleep loss or during several pathologies with associated fatigue, e.g., sleep apnea, rheumatoid arthritis, pre-eclampsia, multiple sclerosis. The proposed experiments seek to understand in mechanistic detail how TNF-alpha is involved in sleep regulation. We will determine whether blocking TNF-alpha or TNF-alpha production centrally attenuates systemic TNF-alpha-induced sleep responses; preliminary data show that vagotomy attenuates systemic TNF-alpha-induced NREMS (Specific Aim #1). We will investigate TNF-alpha regulation of NREMS within specific TNF-active sites in brain (Specific Aim #2). Preliminary data indicate that microinjection of TNF-alpha into the preoptic area enhances NREMS, whereas microinjection of an inhibitor of TNF-alpha reduces NREMS. Pharmacologic blockage of prostaglandins, adenosine, and interleukin-1, and sleep manipulation using sleep deprivation and acute mild increases in ambient temperature to enhance sleep, will be combined with microinjections of TNF-alpha or TNF-alpha inhibitors. We will also use gene arrays to determine the time course of sleep-sensitive changes in brain for TNF and TNF superfamily member mRNAs. Anticipated results will provide molecular-mechanistic advances to understand sleep regulation as well as aid our general understanding of cytokine regulation in the brain. We anticipate that results will be directly relevant to therapeutics, e.g., a TNF soluble receptor has already been shown to reduce fatigue associated with rheumatoid arthritis.